Growth hormone, which is secreted from the pituitary, stimulates growth of all tissues of the body that are capable of growing. In addition, growth hormone is known to have the following basic effects on the metabolic processes of the body: (1) Increased rate of protein synthesis in all cells of the body; (2) Decreased rate of carbohydrate utilization in cells of the body; (3) Increased mobilization of free fatty acids and use of fatty acids for energy. A deficiency in growth hormone secretion can result in various medical disorders, such as dwarfism.
Methodology is known in the art to determine the activity of a compound as a growth hormone secretagogue. For example, an ex vivo assay is described by Smith, et al., Science, 260, 1640–1643 (1993) (see text of FIG. 2 therein), but this assay requires the use of cell cultures and does not give an indication of competitive binding activity. Accordingly, it would be desirable to develop a non-radioactively labeled ligand which can be used to identify and characterize cellular receptors which play a role in the activity of growth hormone secretagogue. It would also be desirable to have a non-radioactively labeled ligand available for use in an assay for testing compounds for growth hormone secretagogue activity.
Such studies normally require a high specific activity radio-ligand. Previous attempts to develop a binding assay using [T]-labeled or [125I]-labeled peptide ligands derived from GHRP-6 met with limited success. See R. F. Walker, et al. Neuropharmacol. 989, 28, 1139 and C. Y. Bowers et al., Biochem. Biophys. Res. Comm. 1991, 178,31 (both of which, to the extent necessary, are herein incorporated by reference). Generally, the binding of such peptide ligands was of low affinity and of excessively high capacity. Moreover, the binding affinities did not correlate with the growth hormone secretory activity of the peptides. The lack of correlation of binding and growth hormone secretory activity most likely was the result of the relatively low specific activity (in the case of [T] GHRP-6) and non-specific binding properties of the radio-ligands.
These problems are solved by the present invention, which provides a fluorescent labeled ghrelin analog (labeled growth hormone secretagogue) which can be used in high throughput screening to identify and characterize cellular receptors which play a role in the activity of growth hormone secretagogue and for use in an assay for testing compounds for growth hormone secretagogue activity.